Sexual contact is a major mode of HIV-1 transmission, with an increasing number of transmissions occurring via heterosexual contact from males to females. Consequently, about 16 million women worldwide are infected with HIV-1. Heterosexual transmission includes both anal and vaginal sex; the estimated risk of infection from vaginal sex with an HIV-1 positive male ranges from 0.08% to 0.30% per sexual act (not including commercial sex workers). This is in contrast with the pooled risk estimate for anal intercourse, which at 1.7% is at least 5?6 times higher than the risk estimate for vaginal sex. These statistics indicate that HIV-1 transmission during vaginal sex faces unique vaginal defenses that prevent the transmission and/or early steps of HIV-1 infection. We have identified vaginal fluid exosomes (VFE) and shown that VFE can significantly reduce HIV-1 transmission in vitro. We hypothesize that VFE can constitute a barrier to HIV-1 transmission during heterosexual contact. To test this hypothesis, we will pursue the following Specific Aims: (1) Analyze VFE and determine their composition and their source; (3) Identify the step(s) of the HIV-1 life cycle that are targeted by VFE; and (3) Determine how VFE block HIV-1 transmission. Specific points of innovation include (1) identification of female-produced defenses against HIV-1 infection, (2) characterization of the newly discovered VFE, and (3) identification of the mechanism by which VFE inhibit HIV-1 transmission. Our research environment enables close collaboration between clinical and basic researchers, which is crucial for the successful outcome of this project.